The invention relates to a method for recognizing resistant germs in a sample material and to a device for performing the method.
A plurality of different bacteria are among the physiological flora of the skin and mucus membranes and thus part of the microbiome, i.e. the entirety of all the microorganisms populating the human body. In populating the human body, the bacteria perform important tasks, such as, for example, protecting the skin and the entire organism from pathogenic germs, i.e. germs dangerous to health, penetrating, since these compete for their habitat, i.e. the human body. When this system loses its balance, for example by a weak immune system, infections may result. Usually, infections caused by bacteria are easy and quick to treat by administering antibiotics.
However, over the years, the pathogens have developed resistances to the active substances used, thereby making therapy considerably more difficult. Germs and pathogens resistant to the active substances used are referred to as multi-resistant pathogens (MRP). This is a big problem in particular in hospitals and in nursing homes, since this is where old and sick people are, whose immune system is weakened and who, consequently, are particularly susceptible to infections. In addition, babies whose immune systems have not yet formed completely and immunosuppressed patients, like those in an intensive care unit, the immune of whom is suppressed by drugs, for example after transplants for minimizing rejection reactions, are particularly at risk of being infected by multi-resistant pathogens.
Worldwide, methicillin-resistant Staphylococcus aureus (MRSA) strains cause most infections of the skin and soft parts contracted in hospitals or nursing homes. Frequently, a sceptic course occurs, which is rated to be particularly critical. Since MRSA is not only resistant to the penicillin methicillin, but also to most other antibiotics routinely administered, therapy here is done using so-called reserve antibiotics. Nevertheless, recovery frequently is very time-consuming and difficult—if successful at all. Apart from the methicillin-resistant Staphylococcus aureus, there are also strains resistant to other antibiotics, such as, for example, vancomycin. Different types of bacteria, such as, for example, enterococci, pneumococci, pseudomonas aeruginosa, campylobacter, EHEC and others, exhibit multiple resistances and consequently are also considered to be problematic germs.
The transmission of MRP takes place either by direct contact of an MRP carrier, for example when using the same bathroom as an MRP carrier, or by treating doctors and nursing staff when neglecting to hygienic measures, in particular strict hand hygiene.
Specific hygiene measures are to be kept in order to minimize the danger of further spreading MRP. Treating doctors and nursing staff wear coats, masks and gloves and thoroughly perform hygienic disinfection of their hands. The patients are to be accommodated in single rooms.
However, in order to take and realize these measures, it is necessitated to recognize the MRP carrier status of a patient already when admitting same to a health institution. It may be advisable here to routinely screen every new patient admitted for MRP. This examination represents an important preventive measure of minimizing and, in the best case, preventing nosocomial infections with multi-resistant pathogens.
In clinical microbiology, examination materials (such as smear test swabs) which carry human-pathogenic germs are spread onto suitable universal and selective media or injected into liquid nutrient broths. After a certain incubation period, colonies may be recognized in an incubator. Cell division in most bacteria takes approximately 20 minutes so that typically days, in some cases (like Mycobacterium tuberculosis) even weeks, may pass until a sufficiently large number of bacteria has grown. The pathogens, however, frequently grow in mixed cultures which subsequently necessitate individual colonies to be isolated so as to obtain pure cultures. These are then multiplied, subjected to microscopy and evaluated morphologically. Using different chromogenic selective media, biochemical detection reactions and special coloring, the germs are identified.
Frequently, characterization of bacteria takes place by identifying a germ-specific enzyme set using color reactions, thereby allowing their determination and classification. Cultivating and characterizing the bacteria takes—depending on the growth behavior of the respective germ—days up to several weeks, as is described in Kayser et al., Brandis et al., and Hallman et al.
Subsequently, an antibiogram is obtained by placing selected, maybe different antibiotics disks on solid nutrient media, like those made of agar, onto which the bacteria have been spread before. Growth inhibition zones on the agar indicate whether and how sensitively the germs react to the corresponding antibiotic of the disks. Usually, an 18 to 24-hour incubation is necessitated here, which, however, may also take considerably longer with certain, mostly demanding pathogens due to slow growth. For a healing process, this may mean that a valuable amount of time elapses until a therapy may be adapted correspondingly. In other words, the antibiotics diffuse into the agar during the 18 to 24-hour incubation. If the pathogen reacts sensitively to an antibiotic, the result will be an inhibition zone around the disk applied.
As an alternative to the time-consuming conventional microbiological cultivation of the pathogens, specific deoxyribonucleic acid (DNA) sequences of multi-resistant Staphylococcus aureus pathogens may also be identified by means of a polymerase chain reaction (PCR). A target sequence to be identified may, for example, be the SCCmec gene. Identifying these pathogen-specific target sequences is mostly done using the classical polymerase chain reaction with corresponding primer sequences. When the DNA sequence which is complementary to the primers is present, it is multiplied. By an interaction of the DNA and the fluorescence dye, like SYBR Green I, the reaction becomes visible and measureable. The more double-strand DNA, the stronger the fluorescence signal.
Alternatively, there are also identifying methods in which identifying the bacteria DNA takes place by hybridizing using fluorescence-labeled target sequences. The Robert-Koch Institute recommends using a nasal/throat smear of the patient as a smear test material. The sensitivity of the MRSA-DNA identification is roughly 95%, specificity 97-99%. Thus, the molecular-biological MRSA direct identification is highly germ-specific and takes about 2-3 hours. However, it is of disadvantage that the molecular-biological identification is, on average, roughly 10 times as expensive as conventional methods and, for practical and organizational reasons, is analyzed only with a time offset after having collected several patient samples concerning the same problem. As experience has shown, this may take place on the next day, but may also take up to several days after having taken the sample. The consequence is that, when admitting the patient to the health institution, there is no result regarding the MRP carrier status and, consequently, it cannot be decided upon at that time whether the patient needs to be isolated or not.
The PCR-based identification is not suitable when checking the therapy course, since the DNA of non-viable bacteria is also identified. A number of living germs or a decrease in the number of living germs, which hints at healing, cannot be detected. Furthermore, this test cannot be used for on-site recognition and is comparably expensive, as is described in Hoffelder et al., Reischl et al., and Warren et al.
Creating a culture for making an antibiogram usually takes place in parallel to performing the molecular-biological examination and takes—depending on the growth behavior of the pathogen—at least one day. In addition, this method may only be performed in special laboratories using high apparatus complexity and done by qualified specialists, thereby making it unsuitable for a quick “bed-side” analysis.
In contrast to PCR methods, there are also antibody-based identification methods for multi-resistant pathogens of which some will be discussed below briefly.
Apart from molecular-biological MRSA screening, bacteriophage-based technologies are also employed. Bacteriophages are viruses which infest bacteria specifically, introduce phage DNA into the host cell, the MRP, use their replication machinery in order to have their virus genome transcribed and have their own proteins produced after translation. The phages multiply within the cell and are set free after cell lysis, i.e. the cell membrane resolving after the cell has died. The proteins produced by the phages may be identified using labeled antibodies and thus allow a specific pathogen identification. This method is also referred to as antibody-based identification method. With this method, advantage is taken from the fact that the multiplication of bacteriophages is considerably faster than that of the bacteria cells themselves, as is described in www.microphage.com
A further strategy for specific MRSA identification is the so-called Q-MAP test, as is described, for example, in www.pcds-gmbh.de. Here, antibodies directed against bacteria-specific antigens are used, which are presented, i.e. arranged, on the surface of the pathogens. The antibodies are bound to magnetic nanoparticles and are incubated with/using the respective sample. When there is an antigen-antibody reaction, micro-precipitates form. These immune complexes may be detected metrologically by a Q-MAP system.
Basically, antibody-based identification strategies are—similarly to the molecular-biological approach—highly specific. However, gaining antibodies is complicated and entails comparatively high manufacturing costs. Since they are proteins and sensitive to temperature, transport and storage necessitate specific conditions. Storage stability is also limited. Reproducibility when manufacturing antibodies in a constant high quality within the individual batches may become problematic, which in turn may result in bottlenecks in the availability.
Creating a culture for obtaining an antibiogram usually takes place in parallel to performing the molecular-biological examination and takes, depending on the growth behavior of the pathogen, at least one day. This means that the specific pathogen identification is only suitable for universal entry screening to a limited extent. Since, in PCR identification, the DNA of non-living pathogens is also detected, this form of identification is not suitable for therapy control, which describes a concentration of living pathogens, for example.
At present, in the routine of health institutions, examinations for recognizing multi-resistant pathogens are done only in suspicious patients. The identification methods are either very time-consuming (microbiological-cultural identification) or very expensive and specific (molecular-biological or antibody-based identification).
Consequently, a quick test for MRP identification which allows a rapid entry examination of patients as to multi-resistant pathogens would be desirable so as to minimize considerably the risk of transmitting the pathogens to other humans and patients.
Consequently, the object underlying the present invention is providing a method and a device implementing the method, allowing multi-resistant pathogens in a sample to be identified at low time requirements.